This invention relates to chemical and biochemical assays, and more particularly to an improved optical apparatus and methods for radiation-emitting assays.
Assays are well known in which aliquots of sample-under-test one or more reagents are variously reacted in highly specific reactions to form ligand/conjugate complexes such as antigen/antibody or similar complexes which may then be observed in order to assay the sample for a titer of a predetermined moiety from the sample. Typically, an antibody is used to assay for the presence of an antigen for which the antibody is specific, but such assays have been extended to quantitate haptens such as hormones, alkaloids, steroids, antigens, antibodies, nucleic acids, and fragments thereof. It is in this broad sense that the term "ligand/conjugate" as used herein should be understood.
Sensitive immunoassays typically use tracer techniques in which a tagged constituent of the complex is incorporated, for example in the reagent, the non-complex tagged reagent then being separated from the complexed reagent. The complex can be thereafter quantitated by observing a signal from the tag. Radioisotopes, fluorescent and chemiluminescent molecules, colorimetric tags, and other radiation-emitting markers have been used to label constituents or moieties of the complex, appropriate apparatus being employed to detect and measure the radiation from the label.
In such assays where at least one component of the conjugate complex is initially bound to a solid substrate preparatory to formation of the complex, a basic problem arises because of the typically lengthy time required to bind that component to the substrate. For example, fluorescent assays such as those performed in the usual 96 well microtiter plate, require time in the order of hours to effect binding of a component to the solid phase, despite such expedients as heating, shaking and the like. The binding delay may be considerably reduced by increasing the surface area of the solid phase made available to binding or coating with a ligand. Flowing the sample through a packed particulate bed speeds reactions between the sample ligand being assayed and a conjugate immobilized on the surface of the particles.
Consequently, in U.S. patent application Ser. No. 07/924,720 filed Aug. 3, 1992, now U.S. Pat. No. 5,372,783, there is disclosed, inter alia, a flow cell for use in apparatus for assaying a fluid sample, the flow cell comprising a hollow, light-transparent conduit through which a fluid sample being assayed can flow. A fluid-porous barrier or screen is emplaced within the conduit so as to define at least one wall of a chamber in which there is disposed a porous mass comprising a plurality of light-transparent particles dimensioned within a specified range of diameters, the particles having immobilized on their surfaces at least a moiety of a ligand/conjugate complex. The porosity of the screen is sufficiently smaller than such range so that the particles are trapped by the screen and the porosity of the mass of transparent particles is selected so as to permit fluid flow of the sample therethrough. The conduit is preferably disposed within a lens so as to extend transversely to the optical axis of the lens through the focal region of the latter.
While this flow cell has been effective to reduce binding delays and speed reaction times between the sample ligand and the immobilized moiety of a ligand/conjugate complex, there is nonetheless an unfortunate tendency for the screen to become plugged with a gelatinous material after several cycles of use with whole blood as the sample, impeding flow and causing assay errors. Changing the material of the screen, for example to relatively non-stick materials such as polytetrafluorethylene, or cleaning the screen with a variety of solvents such as bases, acids, alcohols, detergents and the like did not provide a satisfactory solution.